The present invention relates generally to nanofluidic systems. In particular, the invention provides an architecture including nanovolume crystallization chambers configured for use in x-ray crystallographic diffraction analysis systems. More particularly, the present method and system provides for a low volume crystallization chamber with reduced x-ray scattering suitable for in-situ crystallography. Merely by way of example, the nanofluidic methods and systems described herein have been applied to a chip geometry that supports crystal growth from reduced volumes and features carbon-based elastomers and non-elastomers in layers through which the x-ray beam passes. Although the techniques for nanofluidic systems are applied to x-ray diffraction analysis of macromolecular crystals, it would be recognized that the invention has a much broader range of applicability.
X-ray diffraction of macromolecular crystals is a method relied upon to determine the ternary structure of native proteins, nucleic acids, or protein-ligand complexes. Obtaining useful X-ray diffraction data, however, is complicated by the difficulty of growing diffraction-quality crystals, the mechanical fragility of macromolecular crystals and the accompanying care with which the crystals must be handled, and the innate interference of the device and milieu suspending the macromolecular crystal in the X-ray beam. Current processes for performing all of the steps from crystallization to successful diffraction are complex and time consuming.
Various microvolume apparatus have been developed to screen conditions and reagents for crystallizing proteins from solution. One such apparatus is the TOPAZ® 1.96 screening chip, available from the present assignee, which utilizes microvolume chambers formed in an elastomeric structure that are filled, isolated, and then interfaced to allow combinations of protein solutions to interact with crystallizing reagents through free interface diffusion. Low volume chambers with microfluidic architecture provide for large scale screening of protein-reagent combinations to determine crystallization conditions. Coupled with an automated inspection station (e.g., AutoInspeX®, available from Fluidigm Corporation) multiple conditions are screened with a rapid pace and with minimal operator commitment.
Current methods evaluate the quality of macromolecular crystals through visual assessments made by a person skilled in crystal inspection. Crystals with well defined edges and otherwise of obvious crystalline appearance are typically judged of higher priority than crystals that have visual signs of defects within the lattice of the crystal. Although numerous designs have been advanced to provide for in situ x-ray diffraction of macromolecular crystals, problems are present in these designs. Thus, there is a need in the art for improved methods and systems related to diffraction-based crystallization analysis.